Phosphatidylinositol Transfer Protein-α in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis

Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have reduced intracellular pools of phosphoinositides and an 80% reduction in IP3 generation upon platelet activation. Unexpectedly, mice lacking platelet PITPα form thrombi normally at sites of intravascular injuries. However, following intravenous injection of tumor cells, mice lacking PITPα develop fewer lung metastases due to a reduction of fibrin formation surrounding the tumor cells, rendering the metastases susceptible to mucosal immunity. These findings demonstrate that platelet PITPα-mediated phosphoinositide signaling is inconsequential for in vivo hemostasis, yet is critical for in vivo dissemination. Moreover, this demonstrates that signaling pathways within platelets may be segregated into pathways that are essential for thrombosis formation and pathways that are important for non-hemostatic functions

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins. Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined. Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin wa

Prevalence of Taenia solium cysticercosis in pigs entering the food chain in western Kenya

Three hundred forty-three pigs slaughtered and marketed in western Kenya were subjected to lingual examination and HP10 Ag-ELISA for the serological detection of Taenia solium antigen. When estimates were adjusted for the sensitivity and specificity of the diagnostic assays, prevalence of T. solium cysticercosis estimated by lingual exam and HP10 Ag-ELISA was between 34.4 % (95 % confidence interval (CI) 19.4–49.4 %) and 37.6 % (95 % CI 29.3–45.9 %), respectively. All pigs, however, were reported to have passed routine meat inspection. Since T. solium poses a serious threat to public health, these results, if confirmed, indicate that the introduction of control strategies may be appropriate to ensure the safety of pork production in this region

Genetic Diversity of Dahongjun, the Commercially Important “Big Red Mushroom” from Southern China

BACKGROUND: In southern China, a wild ectomycorrhizal mushroom commonly called "Dahongjun" or "Big Red Mushroom" by the local residents, has been harvested, consumed, and/or exported as an exotic food for many years. Although ecologically and economically important, very little is known about this mushroom, including its diversity and population structure. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we analyzed 122 samples from five local populations representing the known distribution ranges of this mushroom in southern China. We investigated the genetic diversity and geographic structure of this mushroom using sequences from four DNA fragments. Our analyses identified that this mushroom contained at least three divergent lineages: one corresponds to a recently described species Russula griseocarnosa from southern China and the remaining two likely represent two novel species. While these lineages were prominently structured geographically based on ITS sequences, evidence for ancient and/or recent gene flow was also identified within individual lineages. In addition, a local population from Ailaoshan in central Yunnan Province where 85 of our 122 specimens came from showed clear evidence of recombination. CONCLUSION AND SIGNIFICANCE: The ectomycorrhizal mushroom "Dahongjun" from southern China is a species complex with at least three divergent lineages. These lineages are largely geographically structured and there is evidence for recombination in nature. Our results indicate mature Dahongjun mushrooms with abundant basidiospores are important for the reproduction of this mushroom in nature and that individual populations of this species should be managed separately

Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(−) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(−) EB cells showed that CD45(+)Mac-1(−)Ter119(−) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(−)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(−) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(−) and they rapidly acquire CD122 as they differentiate along the NK lineage

Assessment of human cytomegalovirus co-infection in Egyptian chronic HCV patients

Human cytomegalovirus (HCMV) is the most common cause of severe morbidity and mortality in immune- compromised individuals. This study was conducted to determine the incidence of HCMV infection in HCV patients who either spontaneously cleared the virus or progressed to chronic HCV infection. The study included a total of eighty four cases (48 females and 36 males) that were referred to blood banks for blood donation with an age range of 18-64 years (mean age 37.62 ± 10.03 years). Hepatitis C virus RNA and HCMV DNA were detected in sera by RT-nested PCR and nested PCR respectively in all subjects. Immunoglobulin G levels for HCV and HCMV were determined. Besides, IgM antibodies for HCMV infection were also determined in subjects' sera. Fifty three out of 84 cases (63%) were positive for HCV-RNA while 31 (37%) cases had negative HCV RNA. Forty six (87%) and 13 (25%) cases out of 53 HCV RNA positive patients were positive for HCMV IgG and IgM antibodies respectively. While 20 of 53 cases (38%) had detectable HCMV DNA. To examine the role of HCMV infection in HCV spontaneous resolution, two groups of HCV patients, group 1) chronic HCV infection (positive HCV RNA and positive IgG antibodies) vs group 2) spontaneous resolution (negative HCV RNA and positive IgG antibodies) were compared. The percentages of positive CMV IgG and IgM results is higher in chronic HCV patient than those in spontaneously cleared HCV patients and the difference is highly statistically significant (P value < 0.001). Also, there is a general trend towards elevated levels of CMV IgG antibodies in HCV chronic patients than those in spontaneously cleared HCV patients (P value < 0.02). HCMV DNA detection in group 1 was more than twice the value observed in group 2 (38% vs 14.3%, P value < 0.001). Moreover, levels of liver enzymes were significantly higher in HCV RNA positive cases co-infected with HCMV DNA than HCMV negative cases (P value < 0.001). The results indicate the role of HCMV in the liver pathogenesis. We conclude that chronic HCV patients co-infected with HCMV infection can be regarded as high risk groups for liver disease progression where they should be monitored for the long term outcome of the disease

Biochemical Comparison of Anopheles gambiae and Human NADPH P450 Reductases Reveals Different 2′-5′-ADP and FMN Binding Traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (kcat) of 105 s−1 and 88 s−1, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC50 value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (Kd = 410±18 nM) was ∼10 fold weaker than human CPR (Kd = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (Kd = 83±2.0 nM) than the equivalent human domain (Kd = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC50 = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR

A Polymorphism in a Gene Encoding Perilipin 4 Is Associated with Height but not with Bone Measures in Individuals from the Framingham Osteoporosis Study

There is increasing interest in identifying new pathways and candidate genes that confer susceptibility to osteoporosis. There is evidence that adipogenesis and osteogenesis may be related, including a common bone marrow progenitor cell for both adipocytes and osteoblasts. Perilipin 1 (PLIN1) and Perilipin 4 (PLIN4) are members of the PATS family of genes and are involved in lipolysis of intracellular lipid deposits. A previous study reported gender-specific associations between one polymorphism of PLIN1 and bone mineral density (BMD) in a Japanese population. We hypothesized that polymorphisms in PLIN1 and PLIN4 would be associated with bone measures in adult Caucasian participants of the Framingham Osteoporosis Study (FOS). We genotyped 1,206 male and 1,445 female participants of the FOS for four single-nucleotide polymorphism (SNPs) in PLIN1 and seven SNPs in PLIN4 and tested for associations with measures of BMD, bone ultrasound, hip geometry, and height. We found several gender-specific significant associations with the measured traits. The association of PLIN4 SNP rs8887, G>A with height in females trended toward significance after simulation testing (adjusted P = 0.07) and remained significant after simulation testing in the combined-sex model (adjusted P = 0.033). In a large study sample of men and women, we found a significant association between one SNP in PLIN4 and height but not with bone traits, suggesting that PATS family genes are not important in the regulation of bone. Identification of genes that influence human height may lead to a better understanding of the processes involved in growth and development

Merkel cell carcinoma of skin-current controversies and recommendations

The review covers the current recommendations for Merkel cell carcinoma (MCC), with detailed discussion of many controversies. The 2010 AJCC staging system is more in-line with other skin malignancies although more complicated to use. The changes in staging system over time make comparison of studies difficult. A wide excision with margins of 2.5–3 cm is generally recommended. Even for primary </= 1 cm, there is a significant risk of nodal and distant metastases and hence sentinel node biopsy should be done if possible; otherwise adjuvant radiotherapy to the primary and nodal region should be given. Difficulties of setting up trials owing to the rarity of the disease and the mean age of the patient population result in infrequent reports of adjuvant or concurrent chemotherapy in the literature. The benefit, if any, is not great from published studies so far. However, there may be a subgroup of patients with high-risk features, e.g. node-positive and excellent performance status, for whom adjuvant or concurrent chemotherapy may be considered. Since local recurrence and metastases generally occur within 2 years of the initial diagnosis, patients should be followed more frequently in the first 2 years. However delayed recurrence can still occur in a small proportion of patients and long-term follow-up by a specialist is recommended provided that the general condition of the patient allows it. In summary, physician judgment in individual cases of MCC is advisable, to balance the risk of recurrence versus the complications of treatment

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

<p>Abstract</p> <p>Background</p> <p>Immunization of BALB/c mice with a recombinant adenovirus expressing <it>Mycobacterium tuberculosis </it>(<it>M. tuberculosis</it>) antigen 85A (Ad85A) protects against aerosol challenge with <it>M. tuberculosis </it>only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of <it>M. tuberculosis </it>growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.</p> <p>Method</p> <p>Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.</p> <p>Results</p> <p>CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene <it>Cxcr6</it>, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.</p> <p>Conclusions</p> <p>Our microarray analysis represents the first <it>ex vivo </it>study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of <it>M. tuberculosis </it>growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.</p